Proper cell function and avoidance of disease requires subcellular targeting and fusion of transport vesicles with acceptor membranes. The initial recognition between vesicles and acceptor membranes is mediated by peripheral membrane tether protein complexes, and their specific fusion is mediated by integral membrane SNARE complexes. Although the general function of each of these protein families is established, the protein interactions mediating a tethering event-the specific recognition and adhesion of membranes prior to fusion-are largely unknown and our knowledge of the regulatory relationships linking and integrating tether and SNARE function is very rudimentary. Furthermore, intracellular membrane fusion steps are in many cases regulated by and even dependent upon the release of luminal calcium ions, yet documentation of these important roles in specific transport steps, identification of the machinery mediating calcium release and elucidation of the pathways connecting calcium to tether and SNARE function remain elusive. We developed an in vitro reconstitution system for elucidation of tether and SNARE function in the first membrane fusion step in the secretory pathway, the homotypic fusion of COPII vesicles to generate VTCs, a step that was previously unstudied. We will use our in vitro reconstitution in conjunction with in vivo imaging techniques to address the following specific aims: 1) Test the hypothesis that homotypic COPII vesicle tethering is a multi-layered process involving SNARE-signaled tether recruitment and sequential action multiple tether molecules. 2) Test the hypothesis that calcium negatively regulates homotypic COPII vesicle fusion. 3) Test the hypothesis that the luminal calcium concentration intrinsic to each organelle is an important determinant of membrane transport in the early secretory pathway. RELEVANCE TO PUBLIC HEALTH: The homotypic fusion of COPII vesicles produces VTCs, an organelle with a central role in secretory pathway quality control. Multiple diseases, including severe neuropathies, can result from improper VTC trafficking of misfolded secretory proteins, and VTCs have recently been implicated in the biogenesis of viral particles and other functions. A firm understanding of VTC biogenesis is central to an understanding of the role of VTCs in pathogenic states.